Protocol: ADA Bridging ELISA For Use With Anti-Bevacizumab Antibody

This method provides a procedure for generating an ADA ELISA standard curve with anti-bevacizumab antibody, product code HCA185, using bevacizumab antibody for capture and detection. The method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets). This protocol will need to be adjusted for use with different detection methods and immunoassay technology platforms.

Method

Prepare the unconjugated bevacizumab capture antibody at 1 µg/ml in PBS. Coat the required number of wells of a 384-well microtiter plate with 20 µl per well of the prepared bevacizumab. Incubate overnight at 4°C

Wash the microtiter plate five times with PBST.

Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hour at room temperature (RT).

Wash the microtiter plate five times with PBST.

For the standard curve, prepare a dilution series of the anti-bevacizumab antibody HCA185 (AbD17976_hIgG1) in 10% human serum in PBST in triplicate. Final concentration of anti-bevacizumab antibody should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero anti-bevacizumab concentration as the background value.

Add 20 μl of anti-bevacizumab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.

Wash the microtiter plate five times with PBST.

To each well add 20 µl HRP conjugated bevacizumab diluted to 2 µg/ml in HISPEC buffer and incubate for 1 hour at RT.

Wash the microtiter plate ten times with PBST.

Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.